Toxoplasma gondii, a member of the Apicomplexa, is a ubiquitous parasite of mammals. The predilection of this protozoan for the central nervous system causing necrotizing encephalitis constitutes its major presentation in patients with HIV infection. Because it can be transmitted by food or water T. gondii is classified as a category B NIH priority pathogen. During initial infection the rapidly proliferating tachyzoite stage of T. gondii differentiates into the slowly replicating bradyzoite stage that remain latent within tissue cysts for the life of the host; however reactivation of this latent infection can result in disease such chorioretintiis or encephalitis. Several lines of evidence suggest that bradyzoite differentiation is stress mediated and that the cyst wall (a modified parasitophorous vacuole membrane) contains many stage specific proteins and glycoproteins. In the present proposal, studies will be focused on the characterization of the components of the cyst wall. The components of the cyst wall will be characterized using a combination of proteomic, immunologic and genetic approaches taking advantage of techniques we have developed for purification of the T. gondii cyst wall. We have already identified several cyst wall specific proteins that have mucin type domains. Other investigators have previously noted that o-glycosylation occurs in T. gondii and is probably involved in differentiation. We will, therefore, also examine the role of o-glycosylation and mucin type linkages in the development of the cyst wall using biochemical and genetic approaches. Overall these studies will both elucidate the composition of the cyst wall and the glycoproteome of T. gondii providing reagents and insights that should prove useful in understanding the biology and organization of this critical structure. Understanding this structure is important for the development of new strategies to eliminate latent infection and prevent reactivation toxoplasmosis. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is a food and water borne pathogenic organism, an opportunistic infection in AIDS patients and a cause of congenital infection. The cyst wall is a key structure in the pathogenesis of this infection permitting transmission and reactivation. Investigations on the formation of this structure should help define new approaches to control of this pathogenic protozoan. Toxoplasma gondii is a parasite of humans that causes infections resulting in damage to the nervous system. This is due to its ability to form tissue cysts within host cells. The tissue cyst wall is critical for the survival of this organism and its ability to cause latent infection. Many studies, including those from our laboratory group, have suggested that glycoproteins are involved in this process and in the formation of the cyst wall. This proposal will characterize the composition of the cyst wall and its associated glycoproteins. .